Tomato spotted wilt virus (TSWV) causes major losses in vegetable and ornamental crops worldwide. In Australia, lettuce, tomato, and capsicum have been the most severely affected crops in recent years. TSWV is difficult to control because it has an extensive host range of more than 900 plant species including many weeds and it is efficiently transmitted by several thrips species. The DPI Queensland Agricultural Biotechnology Centre (QABC) has developed a highly sensitive and specific molecular diagnostic assay for TSWV with the potential for automation and high throughput while yielding quantitative data on viral load. This project has validated this TSWV assay to provide a more effective test for growers’ samples and to complement and enhance research under way by the national Western Flower Thrips/TSWV projects funded by HAL.